Dynamic chemical modifications to DNA, RNA, and proteins serve as essential regulators of gene expression. Modifications to messenger RNA (mRNA) are purported to regulate RNA stability and structure, translation efficiency, and gene splicing; the most abundant of these modifications is the methylation of the N6 position of adenosine to form N6-methyladenosine (m6A). Significantly, m6A is a major substrate of the fat mass and obesity-associated protein (FTO), and RNA methylation is implicated in a variety of human diseases associated with FTO, including Alzheimer's disease and several types of cancer. However, the specific function of m6A is not currently well understood, as it is exceptionally challenging to identify the modification using traditional RNA sequencing techniques. To fully elucidate the biological role of m6A in mRNA, it is necessary to achieve global mapping of the RNA methylome at single nucleotide resolution. Previous studies have shown that m6A hybridization to canonical adenosine base pairs, thymidine (T) and uridine (U), is thermodynamically destabilized due to unfavorable interactions with the extraneous methyl group. We hypothesize that an unnatural nucleobase that is sterically complementary to m6A can be developed as a sequencing alternative to native base pairs, thereby providing a unique chemical marker for specific m6A sites in RNA. In this proposal, we will synthesize a library of nonstandard nucleobases and identify a complement that can selectively hybridize to m6A using duplex thermal denaturation methods. By employing the principle of steric complementarity in base pairing, we will synthesize a family of unnatural nucleosides derived from 2-pyridone, designed to relieve the steric clashing encountered by the N6-methyl group of m6A and the O4 keto group of T and U. Once a suitable complement to m6A has been identified by thermal denaturation studies, we will incorporate the synthetic base into RNA sequencing methods, first establishing that the complement can be reverse transcribed into cDNA opposite m6A in an RNA template, followed by detection of m6A in native RNA transcripts. This methodology can be applied broadly toward global mapping of the RNA methylome, which will enhance understanding of the role of m6A in cells and, consequently, the implications of aberrant RNA methylation in human disease.